同位素稀释质谱法测定乳及含乳饮料中的酪蛋白含量
目的 建立同位素稀释质谱法对以牛乳、乳粉为原料的饮料中4种不同亚型的酪蛋白进行含量测定。方法 样品溶解后, 经等电点沉淀法提取其中的酪蛋白, 将样品复溶液稀释至适当浓度, 取适量稀释液同时混入多肽同位素内标, 加入胰蛋白酶酶解16 h, 使用甲酸停止酶解反应, 定容后离心, 取上清液分析; 以WATERS ACQUITY UPLC HSS T3 (2.1 mm×50 mm, 1.8 μm) 作分析柱, 用0.3%(V:V)甲酸-水和乙腈作为流动相, 梯度洗脱分离。质谱采用ESI正离子模式检测, 多反应监测模式进行采集, 内标法定量分析。结果 4种不同亚型的酪蛋白特征肽段在4 min内达到基线分离, αs1-酪蛋白、β-酪蛋白、κ-酪蛋白在0.05~8 μmol/L浓度范围内线性良好, αs2-酪蛋白在0.01~5 μmol/L浓度范围内线性良好, 相关系数均在0.9990以上。αs1-酪蛋白、β-酪蛋白、κ-酪蛋白的方法检出限均为3 nmol/L, 定量限均为10 nmol/L, αs2-酪蛋白的方法检出限为1.6 nmol/L, 定量限为5 nmol/L。阴性基质加标的复溶液中, β-酪蛋白浓度在0.5、2.0、5.0 μmol/L 3个水平下的平均回收率为62.21%~84.36%, 相对标准偏差为3.37%~5.50%。采用该方法分别测定了豆奶粉、白咖啡饮品和液体乳饮料中的酪蛋白含量, 结果表明3种样品中均含有4种不同亚型的酪蛋白, 且含量各不相同。结论 该方法灵 敏度高、特异性强, 可以准确快速地测定乳及含乳饮料中酪蛋白含量。
Objective To establish a quantitative method for determination of 4 subtypes of caseins, including αs1-casein, αs2-casein, β-casein and k-casein, in beverage with milk or milk powder as raw materials by isotope dilution mass spectrometry (IDMS). Methods After dissolved, caseins were firstly extracted by isoelectric point method. The precipitated caseins were redissolved in buffer solution, and this solution was diluted to certain concentration. Finally, trypsin was added to the sample diluents which had been mixed with isotope internal peptide control. This enzymolysis reaction was stopped by adding formic acid after 16 h, and then the sample solution was centrifuged after a constant volume and took the supernatant for analysis. The WATERS ACQUITY UPLC HSS T3 (2.1 mm×50 mm, 1.8 μm) was used as the analysis column, and 0.3% (V:V) formate-water and acetonitrile were used as the flow phase for gradient elution and separation. The mass spectra were detected by ESI positive ion model, collected by multi-reaction monitoring model, and analyzed quantitatively by internal standard method. Results Four casein characteristic peptides of different subtypes reached baseline separation within 4 min, αs1-casein, β-casein and κ-casein were all linear in the concentration range of 0.05~8 μmol/L, and αs2-casein was linear in the concentration range of 0.01-5 μmol/L. All the correlation coefficients were greater than 0.9990. The limits of detection (LOD) of αs1-casein, β-casein and κ-casein were 3 nmol/L, and the limits of quantitation (LOQ) of them were 10 nmol/L. The limit of detection of αs2-casein was 1.6 nmol/L, and the limit of quantitation was 5 nmol/L. In the complex solution of negative substrates added, the recoveries of spiked β-casein samples at 3 levels (0.5, 2.0, 5.0 μmol/L) ranged from 62.21% to 84.36%, with relative standard deviations (RSDs) of 3.37%-5.50%. The subtypes and contents of bean milk powder, white coffee drink and milk beverage were measured by this method, and the results showed that all testing samples contained four subtypes of casein, also with different contents. Conclusions This method has high sensitivity and specificity, and can accurately and quickly determine the content of casein in milk and milk beverage.
标题：同位素稀释质谱法测定乳及含乳饮料中的酪蛋白含量
英文标题：Determination of casein in milk and milky beverage by isotope dilution mass spectrometry
作者：
孙姗姗 中国食品药品检定研究院
李婷婷 酒泉市食品检验检测中心
罗娇依 中国食品药品检定研究院;
梁瑞强 中国食品药品检定研究院;
曹进 中国食品药品检定研究院
中文关键词:酪蛋白,同位素稀释,质谱,乳,含乳饮料,
英文关键词:caseins,isotope dilution,mass spectrometry,milk,milky beverage,
发表日期：2018-05-18
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